Protocol for identifying immune checkpoint on circulating tumor cells of human pancreatic ductal adenocarcinoma by single-cell RNA sequencing.

Circulating tumor cells (CTCs) are regarded as the "seeds" of tumor metastasis. Identifying immune checkpoints on CTCs is essential for establishing efficient immunotherapies to prevent tumor metastasis. Here, we present a protocol for isolating CTCs and obtaining single-cell suspensions from pancreatic ductal adenocarcinoma liver metastatic patients. We describe steps for biospecimen acquisition, CTC isolation, and tissue dissociation. We then detail procedures for performing single-cell RNA-seq, annotating cell types, and identifying immune checkpoints on CTCs. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1.

Immune checkpoint HLA-E:CD94-NKG2A mediates evasion of circulating tumor cells from NK cell surveillance.

Circulating tumor cells (CTCs), shed by primary malignancies, function as "seeds" for distant metastasis. However, it is still largely unknown how CTCs escape immune surveillance. Here, we characterize the transcriptomes of human pancreatic ductal adenocarcinoma CTCs, primary, and metastatic lesions at single-cell scale. Cell-interaction analysis and functional studies in vitro and in vivo reveal that CTCs and natural killer (NK) cells interact via the immune checkpoint molecule pair HLA-E:CD94-NKG2A. Disruption of this interaction by blockade of NKG2A or knockdown of HLA-E expression enhances NK-mediated tumor cell killing in vitro and prevents tumor metastasis in vivo. Mechanistic studies indicate that platelet-derived RGS18 promotes the expression of HLA-E through AKT-GSK3ß-CREB signaling, and overexpression of RGS18 facilitates pancreatic tumor hepatic metastasis. In conclusion, platelet-derived RGS18 protects CTCs from NK-mediated immune surveillance by engaging the immune checkpoint HLA-E:CD94-NKG2A. Interruption of the suppressive signaling prevents tumor metastasis in vivo by immune elimination of CTCs.

Loss of MHC-I antigen presentation correlated with immune checkpoint blockade tolerance in MAPK inhibitor-resistant melanoma.

Immune checkpoint blockade and MAPK-targeted combined therapy is a promising regimen for advanced melanoma patients. However, the clinical benefit from this combo regimen remains limited, especially in patients who acquired resistance to MAPK-targeted therapy. Here, we systematically characterized the immune landscape during MAPK-targeted therapy in patients and mouse melanoma models. We observed that both the abundance of tumor-infiltrated T cells and the expression of immune-related genes were upregulated in the drug-responsive period, but downregulated in the resistance period, implying that acquired drug resistance dampens the antitumor immune response. Further transcriptomic dissection indicated that loss of MHC-I antigen presentation on tumor cells plays a critical role in the reduction of T cell infiltration during drug resistance. Survival analysis demonstrates that loss of antigen presentation and reduction of T-cell infiltration during acquired drug resistance are associated with poorer clinical response and prognosis of anti-PD-1 therapy in melanoma patients. In addition, we identified that alterations in the MAPK inhibitor resistance-related oncogenic signaling pathway closely correlated with deficiency of MHC-I antigen presentation, including activation of the PI3K-mTOR, MAPK, and Wnt pathways. In conclusion, our research illuminates that decreased infiltration of T cells is associated with acquired drug resistance during MAPK-targeted therapy, which may underlie the cross-resistance to immune checkpoint blockade.

Dexamethasone for Postoperative Nausea and Vomiting in Papillary Thyroid Carcinoma Patients: A Randomized Clinical Trial.

BACKGROUND: Postoperative nausea and vomiting (PONV) frequently occur after thyroidectomy. Previous studies have investigated the effects of preoperative dexamethasone for alleviating PONV in various cancers, but studies focused on papillary thyroid carcinoma (PTC) were limited. This study aimed to determine the efficacy of a single preoperative dose of dexamethasone to prevent PONV in patients with PTC. METHODS: This single-center, parallel-group, double-blind, placebo-controlled clinical trial was conducted on patients with PTC in West China Hospital. Patients were randomized 1:1 into Group Dex (preoperative 8-mg dexamethasone) or Group Control (0.9% NaCl as control). The primary outcome was the incidence and severity of PONV. The secondary outcomes included postoperative pain, vocal dysfunction, and adverse events. RESULTS: Six hundred participants were recruited and randomized. The total incidence of PONV was 33.3% (200 of 600 patients; 95% CI, 29.6-37.1). In the intention-to-treat analysis, PONV occurred in 81 of 300 patients (27.0%; 95% CI, 21.9-32.1) in Group Dex and in 119 of 300 patients (39.7%; 95% CI, 34.1-45.2) in Group Control (p = 0.001), and the absolute risk reduction was 12.7% (95% CI, 5.1-20.0). Patients in Group Dex reported fewer antiemetic requirements than those in Group Control (p = 0.004). Multivariate analysis indicated that dexamethasone administration (OR = 0.546; 95% CI, 0.383-0.777; p = 0.001) was associated with a reduced rate of PONV. Dexamethasone treatment also contributed to alleviating postoperative pain and improving subjective vocal dysfunction, with no increase in adverse events. CONCLUSIONS: A single dose of dexamethasone is effective and safe for preventing PONV in PTC patients.

Single-cell transcriptomic profiling unravels the adenoma-initiation role of protein tyrosine kinases during colorectal tumorigenesis.

The adenoma-carcinoma sequence is a well-accepted roadmap for the development of sporadic colorectal cancer. However, cellular heterogeneity in aberrant epithelial cells limits our understanding of carcinogenesis in colorectal tissues. Here, we performed a single-cell RNA sequencing survey of 54,788 cells from patient-matched tissue samples, including blood, normal tissue, para-cancer, polyp, and colorectal cancer. At each stage of carcinogenesis, we characterized cell types, transcriptional signatures, and differentially expressed genes of distinct cell populations. The molecular signatures of epithelial cells at normal, benign, and malignant stages were defined at the single-cell scale. Adenoma and carcinoma precursor cell populations were identified and characterized followed by validation with large cohort biopsies. Protein tyrosine kinases (PTKs) BMX and HCK were identified as potential drivers of adenoma initiation. Specific BMX and HCK upregulations were observed in adenoma precursor cell populations from normal and adenoma biopsies. Overexpression of BMX and HCK significantly promoted colorectal epithelial cell proliferation. Importantly, in the organoid culture system, BMX and HCK upregulations resulted in the formation of multilayered polyp-like buds protruding towards the organoid lumen, mimicking the pathological polyp morphology often observed in colorectal cancer. Molecular mechanism analysis revealed that upregulation of BMX or HCK activated the JAK-STAT pathway. In conclusion, our work improved the current knowledge regarding colorectal epithelial evolution during carcinogenesis at the single-cell resolution. These findings may lead to improvements in colorectal cancer diagnosis and treatment.

Combination of MAPK inhibition with photothermal therapy synergistically augments the anti-tumor efficacy of immune checkpoint blockade.

The combination of MAPK-targeted therapy and immune checkpoint blockade is one of the most promising regimens for patients with advanced melanoma. However, the synergistic efficacy of the combo regimen is still controversial in clinical trials. Here, we report that MAPK inhibition induced T-cell suppression within tumor microenvironment is mediated by attenuation of HSP27/HSP70 and deficiency of neoantigen presentation. To address this problem, we designed a photothermal-responsive on-demand controlled drug release gold nano-system to carry BRAF inhibitor. The nano-system can be specifically delivered into tumor cells rather than T-cells, and effectively transformed the optical energy into heat energy upon laser irradiation. Combination of photothermal and targeted therapy significantly promoted immunogenic cell death and T-cell infiltration. On top of this regimen, systematically administration of PD-1 antibody not only suppressed local-treated tumor but also inhibited abscopal tumor by enhancing generalized immune-related antitumor response. More importantly, the triple-combo regimen could efficiently convert immune "cold" tumors into "hot" ones. In conclusion, our research proves the advantage of photothermal-targeted-immune triple combinatorial regimen in treating tumors which are clinical unresectable multifocal and lack of T-cell infiltration.

Improvement of PD-1 Blockade Efficacy and Elimination of Immune-Related Gastrointestinal Adverse Effect by mTOR Inhibitor.

During the past decades, immunotherapy, especially the antibody-mediated immune checkpoint blockade (ICB) has shown durable tumor inhibition and changed the paradigm of cancer treatment. However, a growing body of evidence suggests that ICB treatment induces severe immune-related adverse events (irAEs), and the side effect even leads to the discontinuation of lifesaving treatment. Here, we found that ICB treatment induces colitis in melanoma patients and promotes the infiltration of CD8+ effector T cells into colitic lesions. Further transcriptomic dissection indicated the PI3K-AKT-mTOR pathway was highly activated in CD8+ effector T cells of colitic lesions. Moreover, we developed a mouse melanoma model to recapitulate the gastrointestinal toxicity of anti-PD-1 treatment in clinical settings. Anti-PD-1 treatment significantly contributed to the infiltration of CD8+ T cells, and correspondingly induced severe enteritis. Immunohistochemistry experiments showed that the PI3K-AKT-mTOR pathway of T cells was activated by anti-PD-1 treatment. Blockade of the pathway with mTOR inhibitor sirolimus not only inhibits tumor growth but also suppresses the T cell infiltration in colitic lesions. More importantly, combination with sirolimus and anti-PD-1 synergistically inhibits tumor growth via inducing the immunogenic cell death of tumor cells in vivo. In summary, our research demonstrated the principle of mTOR inhibitor and anti-PD-1 combinatorial therapeutic regimen, which provided a novel therapeutic strategy for irAEs in clinics.

Curcumin-Induced Global Profiling of Transcriptomes in Small Cell Lung Cancer Cells.

BACKGROUND: Curcumin, one of the promising candidates for supplementary therapy in cancer treatment, has been demonstrated by numerous preclinical and clinical evidence to be beneficial in treating various cancers. Apart from the critical role in a deluge of pathological processes, some mRNAs, in particular, microRNAs (miRNAs), are also involved in the anti-tumor activity. Therefore, our research focused on the possible effects of curcumin on small cell lung cancer (SCLC) cells and drew a comprehensive transcriptomes profile by high throughput sequencing to understand the molecular mechanism of curcumin as an anti-tumor agent. METHODS: First, we calculated the apoptosis rate of H446 cells (a human SCLC cell line) cultured with curcumin. The high output sequencing uncovered the altered expression profile of genes and miRNAs. KEGG analysis selected the potential signal pathway associated with the antiproliferative property of curcumin. Finally, miRNAs significantly changed, as well as the regulatory roles of those miRNAs in cell apoptosis were determined. RESULT: The apoptosis rate of H446 cells increased under the elevated concentration of curcumin treatment. And cell cycle-related genes downregulated in the curcumin-treated cells. Besides, miRNA-548ah-5p of a high level acted as a negative role in the anticarcinogenic activity of curcumin. CONCLUSION: Our findings not only enriched the understanding of anti-tumor activity initiated by curcumin through figuring out the downregulated cell cycle-related pathways but also shed light on its novel therapeutic application.

Integrin-Src-YAP1 signaling mediates the melanoma acquired resistance to MAPK and PI3K/mTOR dual targeted therapy.

Activation of PI3K/AKT pathway is one of the most recurrent resistant mechanisms for BRAF-targeted therapy, and the combination of MAPK and PI3K/AKT inhibitors becomes one of the most promising regimens for BRAF-targeted relapsed melanoma patients. Although the potent drug efficacy was observed in preclinical experiments and early clinical trials, the dual-drug resistance is inevitable observed. In this study, we systematically explored the mechanisms of dual-drug resistance to MAPKi and PI3K/mTORi in melanoma. With transcriptomic dissection of dual-drug resistant models, we identified that the drug tolerance was mediated by ECM-integrins α3ß1 and α11ß1 signaling. Upon binding ECM, the integrins activated downstream kinase Src rather than FAK, WNT, or TGFß. Knockdown of integrins α3, α11, and ß1 significantly inhibited the proliferation of dual-drug resistant sublines while with trivial effects on parental cells. Although Src inhibition suppressed the phosphorylation of AKT, c-JUN, and p38, none of inhibitors targeting these kinases reversed the dual-drug resistance in model cells. Notably, Src inhibitor promoted the phosphorylations of LATS1 and YAP1, subsequently, re-localized YAP1 from nucleus to cytosol facilitating further degradation. Both small molecule inhibitors and shRNAs targeting YAP1 or Src overcame the MAPKi and PI3K/mTORi dual-drug resistance. In conclusion, our data not only illuminated an integrin-Src-YAP1 pathway mediated MAPKi and PI3K/mTORi dual-drug resistant mechanism but also provided a potential combinatorial regimen for the drug-relapsed melanoma patients.

Differential diagnosis of pancreatic serous cystadenoma and mucinous cystadenoma: utility of textural features in combination with morphological characteristics.

BACKGROUND: Texture analysis of medical images has been reported to be a reliable method for differential diagnosis of neoplasms. This study was to investigate the performance of textural features and the combined performance of textural features and morphological characteristics in the differential diagnosis of pancreatic serous and mucinous cystadenomas. METHODS: We retrospectively reviewed 59 patients with pancreatic serous cystadenoma and 32 patients with pancreatic mucinous cystadenoma at our hospital. A three-dimensional region of interest (ROI) around the margin of the lesion was drawn manually in the CT images of each patient, and textural parameters were retrieved from the ROI. Textural features were extracted using the LifeX software. The least absolute shrinkage and selection operator (LASSO) method was applied to select the textural features. The differential diagnostic capabilities of morphological features, textural features, and their combination were evaluated using receiver operating characteristic (ROC) analysis, and the area under the receiver operating characteristic curve (AUC) was used as the main indicator. The diagnostic accuracy based on the AUC value is defined as follows: 0.9-1.0, excellent; 0.8-0.9, good; 0.7-0.8, moderate; 0.6-0.7, fair; 0.5-0.6, poor. RESULTS: In the differential diagnosis of pancreatic serous and mucinous cystadenomas, the combination of morphological characteristics and textural features (AUC 0.893, 95% CI 0.816-0.970) is better than morphological characteristics (AUC 0.783, 95% CI 0.665-0.900) or textural features (AUC 0.777, 95% CI 0.673-0.880) alone. CONCLUSIONS: In conclusion, our preliminary results highlighted the potential of CT texture analysis in discriminating pancreatic serous cystadenoma from mucinous cystadenoma. Furthermore, the combination of morphological characteristics and textural features can significantly improve the diagnostic performance, which may provide a reliable method for selecting patients with surgical intervention indications in consideration of the different treatment principles of the two diseases.